The Hepatitis C virus (HCV) is a leading cause of liver disease for which current treatments are inadequate. Worldwide, 170 million are infected with HCV and the disease is projected to kill more people than AIDS within a few years based on a death rate that is likely to triple in the next 20 years. HCV infection of the liver progresses to cirrhosis followed by hepatocellular carcinoma and ultimately liver failure, and is the leading reason for liver transplantation. Such infections have become an increasingly appreciated public health problem, especially because current treatment modalities are inadequate. The current standard of care employs a combination of interferon-a and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus, clearly there is a need and market for better therapies. A critical prerequisite for identifying and developing better therapies for viral hepatitis is the availability of convenient model systems capable of supporting efficient authentic viral replication. Unfortunately, to date the only non-human animal models are the chimpanzee and, with a variety of severe limitations, an immunodeficient xenotransplant mouse model. The great expense, and non-physiologic and low replication levels, respectively, associated with these animal models place great practical limits on their usefulness for rapid and efficient drug discovery and development. The availability of engineered human liver tissue capable of supporting viral infection would be relatively inexpensive, convenient, and ideal for the evaluation of novel antiviral therapies and the study of HCV and other virus-related pathology. This proposal seeks to leverage patented breakthrough technology in the field of engineered, 3-dimensional liver tissue into a novel platform for molecular virology and antiviral development. Keys to the success of this technology include its scalability, reproducibility, and an established ability to yield liver tissues far exceeding previous attempts at organ engineering. We seek to determine whether the engineered tissues can be infected with an efficient hepatitis virus capable of high-level replication and for which highly sensitive and specific detection reagents are available, namely hepatitis delta virus (HDV). We will then extend the potential of this core technology by determining the level of hepatitis C virus (HCV) infection and replication supported by these tissues. Engineered liver tissues will be inoculated with HDV or HCV infectious serum, or transfected with RNA transcribed from an infectious clone. Immunohistochemistry, immunoblot analysis, RNA genome replication by strand-specific northern blots, and de novo produced virus release by serial passaging and quantitative PCR analyses of the media supernatant will determine if infection and replication have occurred. The project detailed in this proposal is designed to test the above hypotheses and translate the results into a valuable new model system for studying viral hepatitis, generating virus for research use and the development of antivirals. Future indications include a unique and invaluable reagent for studying many key aspects of these viral life cycles, their associated pathogenesis, and novel approaches to antiviral therapy, including determination of the infection potential of subsets of hepatic cells, as well as other liver diseases such as non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, hepatic fibrosis, and hepatocellular cancer (HCC).